DESCRIPTION (Applicant's abstract): Recent studies indicate that oxygen centered free radicals are generated in the diaphragm during strenuous contractions and may contribute to the development of muscle fatigue. Previous experiments have been limited, however, to the investigation of either electrically induced fatigue or the examination of limited periods of respiratory loading (e.g., 15 min.). Moreover, the mechanism by which these free radicals are elaborated and the cellular sites altered by reaction with these radicals have not been established. Objective I experiments will examine the respiratory muscles following various regimens of chronic loading for evidence of free radical elaboration, lipid peroxidation and protein oxidation. The investigators will also: a) correlate alterations in indices of free radical generation with alterations in muscle contractile function and muscle histology, b) determine the subcellular distribution of lipid peroxidation within the diaphragm and identify specific proteins altered during sustained loading, and c) determine if loading evokes an upregulation of cellular antioxidant defenses. Objective II experiments will use an isolated rat diaphragmatic preparation to analyze the cellular mechanisms of contraction-induced free radical formation. We will determine whether free radical production is linked to: a) the processes involved in muscle excitation and excitation-contraction coupling, b) influx of calcium into mitochondria during sustained contraction, with a resultant increase in superoxide "leakage" from the electron transport chain, or c) the xanthine oxidase mediated catabolism of hypoxanthine to xanthine with superoxide generation as a byproduct. Objective III experiments will examine the effect of interventions that alter free radical defenses on the evolution of lipid/protein oxidation, respiratory muscle dysfunction and respiratory failure during chronic loading. The preliminary findings suggest that the phenomenon to be investigated in these experiments (i.e., free radical mediated respiratory muscle dysfunction) represents an important factor modulating the evolution of respiratory failure during loaded breathing. The planned experiments should provide important information regarding the pathogenesis of this type of muscle dysfunction and may identify novel approaches to the treatment of patients with lung disease.